Meibomian gland dysfunction is a leading cause of ocular surface disease. However, little is known about the regulatory processes that control the development and maintenance of this sebaceous gland. Here, we identify a novel function for CD147, a transmembrane protein that promotes tissue remodeling through induction of matrix metalloproteinases, in regulating meibocyte differentiation and activity. We found that CD147 localized along basal cells and within discrete membrane domains of differentiated meibocytes in glandular acini containing gelatinolytic activity. Induction of meibocyte differentiation in vitro promoted CD147 clustering and MMP9 secretion, whereas RNAi-mediated abrogation of CD147 impaired MMP9 secretion, concomitant with a reduction in the number of proliferative cells and cytoplasmic lipids. Meibomian glands of CD147 knockout mice had a lower number of acini in both the superior and inferior tarsal plates of the eyelids, and were characterized by loss of lipid-filled meibocytes compared with control mice. Together, our data provide evidence showing that gelatinolytic activity in meibocytes is dependent on CD147, and supports a role for CD147 in maintaining the normal development and function of the meibomian gland.